Process for making thienopyrimidine compounds

ABSTRACT

The invention provides processes of preparing, separating, and purifying PI3K inhibitor, Formula I and II compounds, and novel intermediates for preparing Formula I and II compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/739,434 filed 29Jul. 2010, which is a National Stage Application under 35 U.S.C. §371and claims the benefit of priority to International Application No.PCT/US2008/081204 filed on 24 Oct. 2008, which claims the benefit under35 USC §119(e) of U.S. Provisional Application Ser. No. 60/982,562 filedon 25 Oct. 2007, each of which are incorporated by reference inentirety.

FIELD OF THE INVENTION

The invention relates generally to processes for making and purifyingthienopyrimidine compounds with anti-cancer activity and morespecifically to compounds which inhibit PI3 kinase activity.

BACKGROUND OF THE INVENTION

Phosphatidylinositol (hereinafter abbreviated as “PI”) is one of anumber of phospholipids found in cell membranes. In recent years it hasbecome clear that PI plays an important role in intracellular signaltransduction. Cell signaling via 3′-phosphorylated phosphoinositides hasbeen implicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity(Rameh et al (1999) J. Biol Chem, 274:8347-8350). The enzyme responsiblefor generating these phosphorylated signaling products,phosphatidylinositol 3-kinase (also referred to as PI 3-kinase or PI3K),was originally identified as an activity associated with viraloncoproteins and growth factor receptor tyrosine kinases thatphosphorylate phosphatidylinositol (PI) and its phosphorylatedderivatives at the 3′-hydroxyl of the inositol ring (Panayotou et al(1992) Trends Cell Biol 2:358-60).

Phosphoinositide 3-kinases (PI3K) are lipid kinases that phosphorylatelipids at the 3-hydroxyl residue of an inositol ring (Whitman et al(1988) Nature, 332:664). The 3-phosphorylated phospholipids (PIP3s)generated by PI3-kinases act as second messengers recruiting kinaseswith lipid binding domains (including plekstrin homology (PH) regions),such as Akt and phosphoinositide-dependent kinase-1 (PDK1). Binding ofAkt to membrane PIP3s causes the translocation of Akt to the plasmamembrane, bringing Akt into contact with PDK1, which is responsible foractivating Akt. The tumor-suppressor phosphatase, PTEN, dephosphorylatesPIP3 and therefore acts as a negative regulator of Aid activation. ThePI3-kinases Akt and PDK1 are important in the regulation of manycellular processes including cell cycle regulation, proliferation,survival, apoptosis and motility and are significant components of themolecular mechanisms of diseases such as cancer, diabetes and immuneinflammation (Vivanco et al (2002) Nature Rev. Cancer 2:489; Phillips etal (1998) Cancer 83:41).

The main PI3-kinase isoform in cancer is the Class I PI3-kinase, p110 α(alpha) (U.S. Pat. No. 5,824,492; U.S. Pat. No. 5,846,824; U.S. Pat. No.6,274,327). Other isoforms are implicated in cardiovascular andimmune-inflammatory disease (Workman P (2004) Biochem Soc Trans32:393-396; Patel et al (2004) Proceedings of the American Associationof Cancer Research (Abstract LB-247) 95th Annual Meeting, March 27-31,Orlando, Fla., USA; Ahmadi K and Waterfield Md. (2004) Encyclopedia ofBiological Chemistry (Lennarz W J, Lane M D eds) Elsevier/AcademicPress).

The PI3 kinase/Akt/PTEN pathway is an attractive target for cancer drugdevelopment since such agents would be expected to inhibitproliferation, reverse the repression of apoptosis and surmountresistance to cytotoxic agents in cancer cells. PI3 kinase inhibitorshave been reported (Yaguchi et al (2006) Jour. of the Nat. Cancer Inst.98(8):545-556; U.S. Pat. No. 7,173,029; U.S. Pat. No. 7,037,915; U.S.Pat. No. 6,608,056; U.S. Pat. No. 6,608,053; U.S. Pat. No. 6,838,457;U.S. Pat. No. 6,770,641; U.S. Pat. No. 6,653,320; U.S. Pat. No.6,403,588; U.S. Pat. No. 6,703,414; WO 97/15658; WO 2006/046031; WO2006/046035; WO 2006/046040; WO 2007/042806; WO 2007/042810; WO2004/017950; US 2004/092561; WO 2004/007491; WO 2004/006916; WO2003/037886; US 2003/149074; WO 2003/035618; WO 2003/034997; US2003/158212; EP 1417976; US 2004/053946; JP 2001247477; JP 08175990; JP08176070).

Thienopyrimidine compounds, including Formula I and II compounds, havep110 alpha binding, PI3 kinase inhibitory activity and inhibit thegrowth of cancer cells (WO 2006/046031; US 2008/0039459; US2008/0076768; US 2008/0076758; WO 2008/070740; WO 2008/073785).

Formula I compound, GDC-0941 (Genentech Inc.), is a selective, orallybioavailable inhibitor of PI3K with promising pharmacokinetic andpharmaceutical properties (Folkes et al (2008) Jour. Med. Chem.51:5522-5532; Belvin et al, American Association for Cancer ResearchAnnual Meeting 2008, 99th:April 15, Abstract 4004; Folkes et al,American Association for Cancer Research Annual Meeting 2008, 99th:April14, Abstract LB-146; Friedman et al, American Association for CancerResearch Annual Meeting 2008, 99th:April 14, Abstract LB-110).

Therapeutic combinations of Formula I and II compounds, and certainchemotherapeutic agents are described in “COMBINATIONS OFPHOSPHOINOSITIDE 3-KINASE INHIBITOR COMPOUNDS AND CHEMOTHERAPEUTICAGENTS, AND METHODS OF USE” Belvin et al, filing date 10 Sep. 2008; U.S.Ser. No. 12/208,227.

SUMMARY OF THE INVENTION

An aspect of the invention includes processes of preparing, separating,and purifying compounds of Formulas I and II.

Formula I compound is named as4-(2-(1H-indazol-4-yl)-6-44-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholineand has the structure:

Formula II compound is named as4-(2-(1H-indazol-4-yl)-6-44-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholineand has the structure:

Formula I and II compounds include all stereoisomers, geometric isomers,tautomers, metabolites, and pharmaceutically acceptable salts thereof.Formula I and II compounds are potent inhibitors of PI3K with drug-likephysicochemical and pharmacokinetic properties. Formula I and IIcompounds exhibit selectivity for class Ia PI3Ks over class Ib, inparticular for the P110 alpha subtype (US 2008/0039459; US 2008/0076768;US 2008/0076758).

Another aspect of the invention includes novel intermediates useful forpreparing Formulas I and II compounds including4-chloro-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole 13 and2-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole10

4-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-2-(2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-4-yl)thieno[3,2-d]pyrimidin-4-yl)morpholine14, and THP regioisomer4-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-2-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)thieno[3,2-d]pyrimidin-4-yl)morpholine14A

4-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-2-(2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-4-yl)thieno[2,3-d]pyrimidin-4-yl)morpholine21, and THP regioisomer4-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-2-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)thieno[2,3-d]pyrimidin-4-yl)morpholine21A

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Theinvention is intended to cover all alternatives, modifications, andequivalents which may be included within the scope of the presentinvention. One skilled in the art will recognize many methods andmaterials similar or equivalent to those described herein, which couldbe used in the practice of the present invention. The present inventionis in no way limited to the methods and materials described. In theevent that one or more of the incorporated literature, patents, andsimilar materials differs from or contradicts this application,including but not limited to defined terms, term usage, describedtechniques, or the like, this application controls.

DEFINITIONS

The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of stated features, integers,components, or steps, but they do not preclude the presence or additionof one or more other features, integers, components, steps, or groupsthereof.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand 1 or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or 1 meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counter ion. The counter ion maybe any organic or inorganic moiety that stabilizes the charge on theparent compound. Furthermore, a pharmaceutically acceptable salt mayhave more than one charged atom in its structure. Instances wheremultiple charged atoms are part of the pharmaceutically acceptable saltcan have multiple counter ions. Hence, a pharmaceutically acceptablesalt can have one or more charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.The term “hydrate” refers to the complex where the solvent molecule iswater.

Preparation of Formula I and II Compounds

The Formula I and II compounds of the invention may contain asymmetricor chiral centers, and therefore exist in different stereoisomericforms. It is intended that all stereoisomeric forms of the compounds ofthe invention, including but not limited to, diastereomers, enantiomersand atropisomers, as well as mixtures thereof such as racemic mixtures,form part of the present invention. In addition, the present inventionembraces all geometric and positional isomers. In the structures shownherein, where the stereochemistry of any particular chiral atom is notspecified, then all stereoisomers are contemplated and included as thecompounds of the invention. Where stereochemistry is specified by asolid wedge or dashed line representing a particular configuration, thenthat stereoisomer is so specified and defined.

The compounds of the present invention may exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms.

The compounds of the present invention may also exist in differenttautomeric forms, and all such forms are embraced within the scope ofthe invention. The term “tautomer” or “tautomeric form” refers tostructural isomers of different energies which are interconvertible viaa low energy barrier. For example, proton tautomers (also known asprototropic tautomers) include interconversions via migration of aproton, such as keto-enol and imine-enamine isomerizations. Valencetautomers include interconversions by reorganization of some of thebonding electrons.

The present invention also embraces isotopically-labeled compounds ofthe present invention which are identical to those recited herein, butfor the fact that one or more atoms are replaced by an atom having anatomic mass or mass number different from the atomic mass or mass numberusually found in nature. All isotopes of any particular atom or elementas specified are contemplated within the scope of the compounds of theinvention, and their uses. Exemplary isotopes that can be incorporatedinto compounds of the invention include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine suchas ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³²P, ³³P, ³⁵S, ¹⁸F,³⁶Cl, ¹²³I and ¹²⁵I. Certain isotopically-labeled compounds of thepresent invention (e.g., those labeled with ³H and ¹⁴C) are useful incompound and/or substrate tissue distribution assays. Tritiated (³H) andcarbon-14 (⁴C) isotopes are useful for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Positron emitting isotopes such as ¹⁵O, ¹³N, ¹¹C and ¹⁸Fare useful for positron emission tomography (PET) studies to examinesubstrate receptor occupancy. Isotopically labeled compounds of thepresent invention can generally be prepared by following proceduresanalogous to those disclosed in the Examples herein below, bysubstituting an isotopically labeled reagent for a non-isotopicallylabeled reagent.

Starting materials and reagents for the preparation of Formula I and IIcompounds are generally available from commercial sources such asSigma-Aldrich Chemical (Milwaukee, Wis.) or are readily prepared usingmethods well known to those skilled in the art (e.g., prepared bymethods generally described in Louis F. Fieser and Mary Fieser, Reagentsfor Organic Synthesis, v. 1-19, Wiley, N.Y. (1967-1999 ed.), orBeilsteins Handbuch der organischen Chemie, 4, Aufl. ed.Springer-Verlag, Berlin, including supplements (also available via theBeilstein online database).

The following Schemes 1-8 illustrate the synthesis of Formula I and IIcompounds and certain intermediates and reagents.

Scheme 1 shows the synthesis of4-(2-chlorothieno[3,2-d]pyrimidin-4-yl)morpholine 4 starting bycyclization of methyl 3-amino-thiophenecarboxylate 1 and potassiumcyanate in acetic acid and water at room temperature to givethieno[3,2-d]pyrimidine-2,4(1H,3H)-dione 2. This is an improvement overcyclization of 1 with urea which requires high temperature and evolutionof ammonia gas. Thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione 2 was convertedto 2,4-dichlorothieno[3,2-d]pyrimidine 3 with phosphorous oxychlorideand a catalytic amount of N,N-dimethylaniline (0.75 equiv.) inacetonitrile. Selective substitution at the 4-position with morpholinegave 4.

Cyclization of methyl 3-aminothiophene-2-carboxylate 1 tothieno[3,2-d]pyrimidine-2,4(1H,3H)-dione 2, and methyl2-aminothiophene-3-carboxylate 15 tothieno[2,3-d]pyrimidine-2,4(1H,3H)-dione 16 has previously beenconducted with urea, requiring high temperature and pressure increase byevolution of ammonia gas (Robba, et al (1975) Bulletin de la SocieteChimique de France (3-4, Pt. 2) 587-91). The present invention replacesurea with potassium cyanate to cyclize 1 to 2 (Example 1), andchlorosulfonyl isocyanate to cyclize 15 to 16 (Scheme 6, Example 12).

Scheme 2 shows the synthesis of 4-(methylsulfonyl)piperazin-1-iumchloride 8 starting by N-sulfonylation of1-(tert-butoxycarbonyl)piperazine 6 (BOC-piperazine) withmethanesulfonyl chloride to give tert-butyl4-(methylsulfonyl)piperazine-1-carboxylate 7 which was treated withaqueous hydrogen chloride solution in 1,4-dioxane to give 8.

Scheme 3 shows the synthesis of2-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole10 starting by cyclization of 3-chloro-2-methylaniline 11 with potassiumacetate, acetic anhydride, and isoamyl nitrite to yield4-chloro-1H-indazole 12. The indazole nitrogen of 4-chloro-1H-indazole12 was protected as tetrahydropyranyl (THP) with 3,4-dihydro-2H-pyran,and pyridinium p-toluenesulfonate in dichloromethane to yield4-chloro-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole 13 and a minor amount(about 10%) of the THP regioisomer,4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole. The mixture wasreacted with PdCl₂(PPh₃)₂, tricyclohexylphosphine,bis(pinacolato)diboron, and potassium acetate in DMSO and heated to 130°C. for 16 hours to give 10, containing a minor amount (about 10%) of theTHP regioisomer,1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole.

Scheme 4 shows the synthesis of4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine9 starting with formylation at the 7-position of4-(2-chlorothieno[3,2-d]pyrimidin-4-yl)morpholine 4 (1.0 equiv.) in THFwith n-BuLi in hexanes to give2-chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde 5 afteracidification. Reductive amination of aldehyde 5 was accomplished with4-(methylsulfonyl)piperazin-1-ium chloride 8 and sodium acetate(anhydrous powder) in 1,2-dichloroethane. Trimethyl orthoformate wasadded and stirred for 6 hours, followed by the addition of sodiumtriacetoxyborohydride to give 9.

Scheme 5 shows the synthesis of4-(2-(1H-indazol-4-yl)-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholineI bis mesylate salt by Suzuki coupling of4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine9 in 1,4-dioxane with2-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole10, and bis(triphenylphosphine)palladium (II) chloride in aqueous sodiumcarbonate. The mixture containing the crude THP protected intermediate14, along with a minor amount of THP regioisomer 14A, was concentrated,acetonitrile was added, and the slurry was filtered. The resulting cakewas dried to afford 14 as a brown-yellow solid with residual Pd contentof 2000 ppm. The cake was dissolved in methylene chloride and FLORISIL®(60-100 mesh, Sigma-Aldrich Chemical Company, Inc) as a palladiumscavenger was then added. FLORISIL® (U.S. Silica Company) is a magnesiumsilicate, highly selective adsorbent.

The slurry was stirred at ambient temperature for a minimum of 5 hours,then SILIABOUND®Thiol (Silicycle Inc) was added. After a minimum of 12hour agitation, the mixture was filtered and rinsed with methylenechloride and ethyl acetate. The filtrate and the rinse were concentratedto give 14 as an off-white solid with Pd content of less than 20 ppm.

4-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-2-(2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-4-yl)thieno[3,2-d]pyrimidin-4-yl)morpholine14 was dissolved in a mixture of methanol and water. Methanesulfonicacid was slowly added and the slurry was stirred at ambient temperaturefor 1 hour, then heated to 65° C. and stirred for 16 hours to afford Ias the bis mesylate salt. The salt was then recrystallized in a mixtureof water and methanol in the presence of additional methanesulfonicacid.

A variety of palladium catalysts can be used during the Suzuki couplingstep to form compounds 14 and 21. Suzuki coupling is a palladiummediated cross coupling reaction of an arylhalide, such as 9 and 20,with a boronic acid such as 10. Low valent, Pd(II) and Pd(0) catalystsmay be used to prepare 14 and 21, including PdC12(PPh₃)₂, Pd(t-Bu)₃,PdCl₂ dppf CH₂Cl₂, Pd(PPh₃)₄, Pd(OAc)/PPh₃, Cl₂Pd[(Pet₃)]₂, Pd(DIPHOS)₂,Cl₂Pd(Bipy), [PdCl(Ph₂PCH₂PPh₂)]₂, Cl₂Pd[P(o-tol)₃]₂,Pd₂(dba)₃/P(o-tol)₃, Pd₂(dba)/P(furyl)₃, Cl₂Pd[P(furyl)₃]₂,Cl₂Pd(PMePh₂)₂, Cl₂Pd[P(4-F-Ph)₃]₂, Cl₂Pd[P(C₆F₆)₃]₂,Cl₂Pd[P(2-COOH-Ph)(Ph)₂]₂, Cl₂Pd[P(4-COOH-Ph)(Ph)₂]₂, and encapsulatedcatalysts Pd EnCat™ 30, Pd EnCat™ TPP30, and Pd(II)EnCat™ BINAP30 (US2004/0254066).

A variety of solid adsorbent palladium scavengers can be used to removepalladium after the Suzuki coupling step to form compounds 14 and 21.Exemplary embodiments of palladium scavengers described herein (Examples10 and 17) include FLORISIL®, SILIABOUND®Thiol, and SILIABOND® Thiourea.Other palladium scavengers include silica gel, controlled-pore glass(TosoHaas), and derivatized low crosslinked polystyrene QuadraPure™ AEA,QuadraPure™ IMDAZ, QuadraPure™ MPA, QuadraPure™ TU (Reaxa Ltd.,Sigma-Aldrich Chemical Co.).

Scheme 6 shows the synthesis of4-(2-chlorothieno[2,3-d]pyrimidin-4-yl)morpholine 18 starting bycyclization of methyl 2-amino-thiophenecarboxylate 15 (95 g) withchlorosulfonyl isocyanate at low temperature remains (−60° C. to −55°C.) to give thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione 16. Phosphorousoxychloride was added slowly to a cold solution ofthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione 16 and N,N-dimethylaniline(0.75 equiv.) in acetonitrile while maintaining the temperature below25° C. The mixture was then heated to 80-85° C. and stirred for 24 hoursto afford dichlorothieno[2,3-d]pyrimidine 17. Morpholine (2.2 equiv.)was added to a solution of 2,4-dichlorothieno[2,3-d]pyrimidine 17 inmethanol and stirred at ambient temperature for 1 h to give 18.

Scheme 7 shows the synthesis of4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholine20 by formylation of 4-(2-chlorothieno[2,3-d]pyrimidin-4-yl)morpholine18 in THF at −78° C. with n-BuLi in hexanes (1.2 equiv.). The resultingslurry was allowed to warm up to −60° C., cooled to −78° C. and DMF (1.5equiv.) was added slowly to afford2-chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbaldehyde 19. To asuspension of 19, 4-(methylsulfonyl)piperazin-1-ium chloride 8(alternatively named as 1-(methylsulfonyl)piperazine hydrochloride, 1.45equiv.) and anhydrous sodium acetate in 1,2-dichloroethane was addedtrimethyl orthoformate (10 equiv.). The slurry was stirred at ambienttemperature for at least 6 hours, then sodium triacetoxyborohydride wasadded and the reaction was stirred for 24 hours to give 20.

Scheme 8 shows the synthesis of4-(2-(1H-Indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholineII sulfate salt starting with Suzuki coupling of4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholine20 in 1,4-dioxane with2-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole10 (1.25 equiv.) and bis(triphenylphosphine)palladium (II) chloride(0.02 equiv.) in aqueous sodium carbonate. The mixture was heated to 88°C. and stirred for 14 hours. The reaction mixture was cooled, filtered,rinsed with water, and stirred with FLORISIL® (60-100 mesh,Sigma-Aldrich Chemical Company, Inc) in methylene chloride at ambienttemperature for 5 hours. The mixture was filtered, rinsed with methylenechloride and ethyl acetate, and the filtrate and the rinse were combinedand concentrated to give solid 21 with Pd content of 150 ppm. The solidwas dissolved in methylene chloride and SILIABOND® Thiourea (SilicycleInc) was added. The mixture was stirred for 5 hours, filtered, rinsedwith methylene chloride and ethyl acetate. All the filtrate and therinse were combined and concentrated to give4-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-2-(2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-4-yl)thieno[2,3-d]pyrimidin-4-yl)morpholine21 as a solid in 70% yield with Pd content<10 ppm), along with a minoramount of THP regioisomer 21A.

THP protected 21 was mixed with methanol and water, cooled to 0° C. anda cold solution of aqueous sulfuric acid (1.20 equiv.) was slowly addedwhile maintaining the temperature below 10° C. The mixture was allowedto warm up to ambient temperature and stirred for 20 hours. The slurrywas cooled to 5° C., filtered and rinsed with cold methanol. The cakewas stirred in aqueous methanol at 50° C. for 3 hours, then cooled 0-5°C., filtered and rinsed with cold methanol to give a cake which wasdried in a vacuum oven to afford II as a light yellow solid sulfate saltin 94% yield.

Methods of Separation

In the methods of preparing the compounds of this invention, it may beadvantageous to separate reaction products from one another and/or fromstarting materials. The desired products of each step or series of stepsis separated and/or purified (hereinafter separated) to the desireddegree of homogeneity by the techniques common in the art. Typicallysuch separations involve multiphase extraction, crystallization from asolvent or solvent mixture, distillation, sublimation, orchromatography. Chromatography can involve any number of methodsincluding, for example: reverse-phase and normal phase; size exclusion;ion exchange; high, medium and low pressure liquid chromatographymethods and apparatus; small scale analytical; simulated moving bed(SMB) and preparative thin or thick layer chromatography, as well astechniques of small scale thin layer and flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like.

Selection of appropriate methods of separation depends on the nature ofthe materials involved. For example, boiling point and molecular weightin distillation and sublimation, presence or absence of polar functionalgroups in chromatography, stability of materials in acidic and basicmedia in multiphase extraction, and the like. One skilled in the artwill apply techniques most likely to achieve the desired separation.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present invention may be atropisomers(e.g., substituted biaryls) and are considered as part of thisinvention. Enantiomers can also be separated by use of a chiral HPLCcolumn.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S. “Stereochemistry of OrganicCompounds,” John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,(1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of chiralcompounds of the invention can be separated and isolated by any suitablemethod, including: (1) formation of ionic, diastereomeric salts withchiral compounds and separation by fractional crystallization or othermethods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: “DrugStereochemistry, Analytical Methods and Pharmacology,” Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-phenylethylamine (amphetamine), and the like withasymmetric compounds bearing acidic functionality, such as carboxylicacid and sulfonic acid. The diastereomeric salts may be induced toseparate by fractional crystallization or ionic chromatography. Forseparation of the optical isomers of amino compounds, addition of chiralcarboxylic or sulfonic acids, such as camphorsulfonic acid, tartaricacid, mandelic acid, or lactic acid can result in formation of thediastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(E. and Wilen, S. “Stereochemistry of Organic Compounds”, John Wiley &Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed byreacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the pure orenriched enantiomer. A method of determining optical purity involvesmaking chiral esters, such as a menthyl ester, e.g., (−) menthylchloroformate in the presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem.,(1982) 47:4165), of the racemic mixture, and analyzing the ¹H NMRspectrum for the presence of the two atropisomeric enantiomers ordiastereomers. Stable diastereomers of atropisomeric compounds can beseparated and isolated by normal- and reverse-phase chromatographyfollowing methods for separation of atropisomeric naphthyl-isoquinolines(WO 96/15111). By method (3), a racemic mixture of two enantiomers canbe separated by chromatography using a chiral stationary phase (“ChiralLiquid Chromatography” (1989) W. J. Lough, Ed., Chapman and Hall, NewYork; Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purifiedenantiomers can be distinguished by methods used to distinguish otherchiral molecules with asymmetric carbon atoms, such as optical rotationand circular dichroism.

Pharmaceutical Formulations

In order to use a compound of this invention for the therapeutictreatment (including prophylactic treatment) of mammals includinghumans, it is normally formulated in accordance with standardpharmaceutical practice as a pharmaceutical composition. According tothis aspect of the invention there is provided a pharmaceuticalcomposition comprising a compound of this invention in association witha pharmaceutically acceptable diluent or carrier.

A typical formulation is prepared by mixing a compound of the presentinvention and a carrier, diluent or excipient. Suitable carriers,diluents and excipients are well known to those skilled in the art andinclude materials such as carbohydrates, waxes, water soluble and/orswellable polymers, hydrophilic or hydrophobic materials, gelatin, oils,solvents, water and the like. The particular carrier, diluent orexcipient used will depend upon the means and purpose for which thecompound of the present invention is being applied. Solvents aregenerally selected based on solvents recognized by persons skilled inthe art as safe (GRAS) to be administered to a mammal. In general, safesolvents are non-toxic aqueous solvents such as water and othernon-toxic solvents that are soluble or miscible in water. Suitableaqueous solvents include water, ethanol, propylene glycol, polyethyleneglycols (e.g., PEG 400, PEG 300), etc. and mixtures thereof. Theformulations may also include one or more buffers, stabilizing agents,surfactants, wetting agents, lubricating agents, emulsifiers, suspendingagents, preservatives, antioxidants, opaquing agents, glidants,processing aids, colorants, sweeteners, perfuming agents, flavoringagents and other known additives to provide an elegant presentation ofthe drug (i.e., a compound of the present invention or pharmaceuticalcomposition thereof) or aid in the manufacturing of the pharmaceuticalproduct (i.e., medicament).

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration. Forexample, a compound of Formula I or II having the desired degree ofpurity may optionally be mixed with pharmaceutically acceptablediluents, carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.), in the formof a lyophilized formulation, milled powder, or an aqueous solution.Formulation may be conducted by mixing at ambient temperature at theappropriate pH, and at the desired degree of purity, withphysiologically acceptable carriers, i.e., carriers that are non-toxicto recipients at the dosages and concentrations employed. The pH of theformulation depends mainly on the particular use and the concentrationof compound, but may range from about 3 to about 8. Formulation in anacetate buffer at pH 5 is a suitable embodiment. The compound of thisinvention for use herein is preferably sterile. In particular,formulations to be used for in vivo administration must be sterile. Suchsterilization is readily accomplished by filtration through sterilefiltration membranes. The compound ordinarily can be stored as a solidcomposition, a lyophilized formulation or as an aqueous solution.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theactive pharmaceutical ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations of Formula I and II compounds may beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing aFormula I or II compound, which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate) and poly-D-(−)-3-hydroxybutyric acid.

The formulations include those suitable for the administration routesdetailed herein. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Techniques and formulations generally are found inRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).Such methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations of a Formula I or II compound suitable for oraladministration may be prepared as discrete units such as pills,capsules, cachets or tablets each containing a predetermined amount of aFormula I or II compound.

Compressed tablets may be prepared by compressing in a suitable machinethe active ingredient in a free-flowing form such as a powder orgranules, optionally mixed with a binder, lubricant, inert diluent,preservative, surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and optionally are formulated so as toprovide slow or controlled release of the active ingredient therefrom.

Tablets, troches, lozenges, aqueous or oil suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, e.g., gelatincapsules, syrups or elixirs may be prepared for oral use. Formulationsof compounds of Formula I or II intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents including sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide a palatablepreparation. Tablets containing the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipient which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium or sodium carbonate, lactose,calcium or sodium phosphate; granulating and disintegrating agents, suchas maize starch, or alginic acid; binding agents, such as starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc. Tablets may be uncoated or may be coated by knowntechniques including microencapsulation to delay disintegration andadsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

Aqueous suspensions of Formula I or II compounds contain the activematerials in admixture with excipients suitable for the manufacture ofaqueous suspensions. Such excipients include a suspending agent, such assodium carboxymethylcellulose, croscarmellose, povidone,methylcellulose, hydroxypropyl methylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally occurring phosphatide (e.g.,lecithin), a condensation product of an alkylene oxide with a fatty acid(e.g., polyoxyethylene stearate), a condensation product of ethyleneoxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

The pharmaceutical compositions of compounds of Formula I or II may bein the form of a sterile injectable preparation, such as a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, such as a solution in 1,3-butanediol or prepared as alyophilized powder. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile fixed oils may conventionally be employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid may likewise be used in the preparationof injectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to 20% w/w, for exampleabout 0.5 to 10% w/w, for example about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

EXAMPLES

In order to illustrate the invention, the following examples areincluded. However, it is to be understood that these examples do notlimit the invention and are only meant to suggest a method of practicingthe invention. Persons skilled in the art will recognize that thechemical reactions described may be readily adapted to alternativemethods for preparing the compounds of this invention which are deemedto be within the scope of this invention.

In the examples described below, unless otherwise indicated alltemperatures are set forth in degrees Celsius (° C.). Reagents werepurchased from commercial suppliers such as Sigma-Aldrich ChemicalCompany, and were used without further purification unless otherwiseindicated.

The reactions set forth below were done generally under a positivepressure of nitrogen or argon or with a drying tube (unless otherwisestated) in anhydrous solvents, and the reaction flasks were typicallyfitted with rubber septa for the introduction of substrates and reagentsvia syringe. Glassware was oven dried and/or heat dried.

Column chromatography was conducted on a Biotage system (Manufacturer:Dyax Corporation) having a silica gel column or on a silica SEP PAK®cartridge (Waters). ¹H NMR spectra were obtained in deuterated CDCl₃,d₆-DMSO, CH₃OD or d₆-acetone solutions (reported in ppm), usingchloroform as the reference standard (7.25 ppm). When peakmultiplicities are reported, the following abbreviations are used: s(singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd(doublet of doublets), dt (doublet of triplets). Coupling constants,when given, are reported in Hertz (Hz).

Example 1 Thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione 2

To a mixture of methyl 3-amino-thiophenecarboxylate 1 (850 g, 5.41 mol,1.0 equiv.), acetic acid (6 L) and water (5 L), a solution of potassiumcyanate (KOCN, 1316 g, 16.22 mole, 3.0 equiv.) in water (3.2 L) wasadded slowly over a period of 1 hour. The resulting mixture was stirredat the ambient temperature for 20 hour, filtered and rinsed with water(4 L). The cake was charged to a suitably sized reactor and 2 M aqueoussodium hydroxide solution (14 L) was added. The slurry was stirred for 2hours and LCMS confirmed the formation of the desired product. Themixture was cooled to 10° C. and 3 M aqueous hydrochloric acid (˜11 L)was added until pH=5.0-6.0 (by pH paper). The slurry was filtered,rinsed with water (6 L), dried in vacuum oven at 50° C. for 24 hours toafford thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione 2 as an off-white solid(834 g, 92%). ¹H NMR (400 MHz, DMSO-d₆) δ 6.90 (d, J=5.2 Hz, 1H), 8.10(d, J=5.2 Hz, 1H), 5.40-5.55 (br s, 2H). LCMS (ESI pos) m/e 169 (M+1).

Example 2 2,4-Dichlorothieno[3,2-d]pyrimidine 3

Phosphorous oxychloride (299 ml, 3.27 mol, 5.0 equiv.) was added slowlyto a cold solution of thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione 2 (110 g,0.654 mol, 1.0 equiv.) and N,N-dimethylaniline (62 ml, 0.491 mol, 0.75equiv.) in acetonitrile (550 ml) while maintaining the temperature below20° C. The mixture was then heated to 80-85° C. and stirred for 24hours. LCMS indicated that the reaction was complete. The reactionmixture was cooled to 15° C., and then poured slowly onto a mixture ofice and cold water (1.0 L). The resulting slurry was filtered, rinsedwith cold water (300 ml). The cake was dried in vacuum oven at 40° C.for 24 hours to afford 2,4-dichlorothieno[3,2-d]pyrimidine 3 as anoff-white solid (93.4 g, 67% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.56(d, J=5.5 Hz, 1H), 8.76 (d, J=5.5 Hz, 1H). LCMS (ESI pos) m/e 205 (M+1).

Example 3 4-(2-Chlorothieno[3,2-d]pyrimidin-4-yl)morpholine 4

Morpholine (87 ml, 1.00 mol, 2.2 equiv.) was added to a solution of2,4-dichloro-thieno[3,2-d]pyrimidine 3 (93.4 g, 0.456 mol, 1.0 equiv.).The reaction mixture was stirred at ambient temperature for 1 h and theresulting slurry was filtered, rinsed with water (500 ml). The cake wasdried in a vacuum oven at 40° C. for 24 hours to give4-(2-chlorothieno[3,2-d]pyrimidin-4-yl)morpholine 4 as an off-whitesolid (109 g, 94% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 3.74 (t, J=4.9 Hz,4H), 3.90 (t, J=4.9 Hz, 4H), 7.40 (d, J=5.6 Hz, 1H), 8.30 (d, J=5.6 Hz,1H). LCMS (ESI pos) m/e 257 (M+1).

Example 4 2-Chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde

To a suspension of 4-(2-chlorothieno[3,2-d]pyrimidin-4-yl)morpholine 4(50 g, 195 mmol, 1.0 equiv.) in THF (anhydrous, 800 ml) at −78° C. wasadded slowly 2.5 M solution of n-BuLi in hexanes (93.9 ml, 234.6 mmol,1.2 equiv.). The resulting slurry was allowed to warm up to −60° C. anda clear brown solution was observed. The solution was then cooled to−78° C. and DMF (anhydrous, 22.7 ml, 293 mmol, 1.5 equiv.) was addedslowly. The resulting solution was stirred at −78° C. for 0.5 hour, thenwarmed up slowly to 0° C. over a period of 1-1.5 hours. The solution wasthen poured slowly to a mixture of 0.25 M aq. hydrochloric acid (1.65 L)and ice water (800 ml). The resulting slurry was stirred at 0-10° C. for0.5 hour, filtered and rinsed with cold water (200 ml). The cake wasdried in vacuum oven at 40° C. for 24 hours to afford2-chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde 5 as a lightyellow solid (54.9 g, 99% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 3.76 (t,J=4.9 Hz, 1H), 3.95 (t, J=4.9 Hz, 4H), 8.28 (s, 1H), 10.20 (s, 1H). LCMS(ESI pos) m/e 285 (M+1).

Example 5 4-(Methylsulfonyl)piperazin-1-ium chloride 8

Methanesulfonyl chloride (34.38 ml, 443 mmol, 1.1 equiv.) was addedslowly to a solution of 1-(tert-butoxycarbonyl)piperazine 6(BOC-piperazine, 75 g, 403 mmol, 1.0 equiv.) and triethylamine (67.4 ml,483 mmol, 1.2 equiv.) in methylene chloride (750 ml) while maintainingthe internal temperature below 20° C. The solution was stirred atambient temperature for 24 hours. The solution was poured onto a mixtureof ice and water (1.5 L). The phases were separated and the aq. phasewas extracted with methylene chloride (800 ml×2). The organic phaseswere combined, dried over MgSO₄, filtered and concentrated to givetert-butyl 4-(methylsulfonyl)piperazine-1-carboxylate 7 as an off-whitesolid (105 g).

¹H NMR (400 MHz, CDCl₃) δ 1.45 (s, 9H), 2.75 (s, 3H), 3.15 (m, 4H), 3.50(m, 4H).

A 4M hydrogen chloride solution in 1,4-dioxane (1.2 L) was added slowlyto a cold solution of tert-butyl4-(methylsulfonyl)piperazine-1-carboxylate 7 (105 g) in methylenechloride (1.1 L) while maintaining the internal temperature below 20° C.The solution was stirred for 20 hours and ¹H NMR indicated that thereaction was complete. The resulting slurry was filtered and rinsed withmethylene chloride (300 ml). The cake was dried in a vacuum oven at 50°C. for 20 hours to afford 4-(methylsulfonyl)piperazin-1-ium chloride 8as a white solid (78.4 g, 97% yield over 2 steps). ¹H NMR (400 MHz,CDCl₃) δ 3.00 (s, 3H), 3.17 (m, 4H), 3.38 (m, 4H), 9.45 (br s, 2H).

Example 64-(2-Chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine9

To a suspension of2-chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde 5 (20.6 g,72.6 mmol, 1.0 equiv.), 4-(methylsulfonyl)piperazin-1-ium chloride 8(18.9 g, 94.4 mmol, 1.30 equiv.) and sodium acetate (anhydrous powder,7.74 g, 94.4 mmol, 1.30 equiv.) in 1,2-dichloroethane (anhydrous, 412ml) was added trimethyl orthoformate (79.5 ml, 726 mmol, 10 equiv.). Theslurry was stirred at ambient temperature for at least 6 hours. Sodiumtriacetoxyborohydride (assay>90%, 20.5 g, 87.1 mmol, 1.2 equiv.) wasadded and the reaction was stirred for 24 hours. LC/MS indicated thatthe reaction was complete. The reaction was quenched with water (1.0 L)and methylene chloride (1.0 L). The phases were separated and theorganic phase was dried over MgSO₄, filtered and concentrated to give ayellow solid (35 g). The crude solid was then stirred in ethyl acetate(500 ml) at 80° C. for 2 hours. The slurry was cooled to 30-40° C.,filtered and rinsed with ethyl acetate (50 ml). The cake was dried invacuum oven at 45° C. to give4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine9 as an off-white solid (24 g, 75% yield). ¹H NMR (400 MHz, DMSO-d₆) δ2.53-2.60 (m, 4H), 2.90 (s, 3H), 3.09-3.19 (m, 4H), 3.73 (t, J=4 Hz,4H), 3.89 (t, J=4 Hz, 4H), 3.91 (s, 2H), 7.31 (s, 1H). LCMS (ESI pos)m/e 432 (M+1).

Example 7 4-Chloro-1H-indazole 12

To a 250 ml flask with stir bar was added 3-chloro-2-methylaniline 11(8.4 ml, 9.95 g, 70.6 mmol), potassium acetate (8.3 g, 84.7 mmol) andchloroform (120 ml). This mixture was cooled to 0° C. with stirring. Tothe cooled mixture was added acetic anhydride (20.0 ml, 212 mmol) dropwise over 2 minutes. The reaction mixture was warmed to 25° C. andstirred for 1 hour. At this point, the reaction was heated to 60° C.Isoamyl nitrite (18.9 ml, 141 mmol) was added and the reaction wasstirred overnight at 60° C. Once complete, water (75 ml) and THF (150ml) were added and the reaction was cooled to 0° C. Lithium hydroxide(LiOH, 20.7 g, 494 mmol) was added and the reaction was stirred at 0° C.for 3 hours. Water (200 ml) was added and the product was extracted withEtOAc (300 ml, 100 ml). The organic layers were combined, dried withMgSO₄ and concentrated to yield 4-chloro-1H-indazole 12 as an orangesolid (11.07 g (100%). ¹H NMR (400 MHz, CDCl₃) δ 8.18 (d, J=1 Hz, 1H),7.33 (d, J=8 Hz 1H), 7.31 (t, J=7 Hz, 1H), 7.17 (dd, J=7 Hz, 1 Hz 1H).LCMS (ESI pos) m/e 153 (M+1).

Example 8 4-Chloro-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole 13

To a 1 L flask with mechanical stirrer was added 4-chloro-1H-indazole 12(75.0 g, 0.492 mol), pyridinium p-toluenesulfonate (1.24 g, 4.92 mmol),CH₂Cl₂ (500 ml) and 3,4-dihydro-2H-pyran (98.6 ml, 1.08 mol). Withstirring, this mixture was heated to 45° C. for 16 hours. Analysis ofreaction mixture shows production of both isomers of product. Cooledreaction to 25° C. and added CH₂Cl₂ (200 ml). Washed the solution withwater (300 ml) and saturated NaHCO₃ (250 ml). Dried the organics withMgSO₄ and concentrated to dryness. Purified the crude product bydissolving in EtOAc/Hexanes (4:6, 1 L) and adding SiO₂ (1.2 L). Themixture was filtered and the cake was washed with EtOAc/Hexanes (4:6, 2L). The organics were concentrated to yield4-chloro-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole 13 as an orange solid(110.2 g, 95%) and a minor amount (about 10%) of the THP regioisomer,4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole. Isomer 1: ¹H NMR (400MHz, CDCl₃) δ 8.10 (d, J=1 Hz, 1H), 7.50 (dd, J=9 Hz, 1 Hz 1H), 7.29(dd, J=9 Hz, 8 Hz 1H), 7.15 (dd, J=8 Hz, 1 Hz 1H) 5.71 (dd, J=9 Hz, 3 Hz1H) 4.02 (m, 1H) 3.55 (m, 1H) 2.51 (m, 1H) 2.02 (m, 2H) 1.55 (m, 3H).LCMS (ESI pos) m/e 237 (M+1); Isomer 2: ¹H NMR (400 MHz, CDCl₃) δ 8.25(d, J=1 Hz, 1H), 7.62 (dd, J=9 Hz, 1 Hz 1H), 7.20 (dd, J=9 Hz, 8 Hz 1H),7.06 (dd, J=8 Hz, 1 Hz 1H) 5.69 (dd, J=9 Hz, 3 Hz 1H) 4.15 (m, 1H) 3.80(m, 1H) 2.22 (m, 2H) 2.05 (m, 1H) 1.75 (m, 3H). LCMS (ESI pos) m/e 237(M+1).

Example 92-(Tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole10

To a 500 ml flask with stir bar was added4-chloro-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole 13 (10.0 g, 42.2mmol), DMSO (176 ml), PdCl₂(PPh₃)₂ (6.2 g, 8.86 mmol),tricyclohexylphosphine (0.47 g, 1.69 mmol), bis(pinacolato)diboron (16.1g, 63.4 mmol) and potassium acetate (12.4 g, 0.127 mol). With stirring,the mixture was heated to 130° C. for 16 hours. The reaction was cooledto 25° C. and EtOAc (600 ml) was added and washed with water (2×250 ml).The organics were dried with MgSO₄ and concentrated to dryness. Thecrude product was purified by SiO₂ plug (120 g), eluting with 10%EtOAc/Hexanes (1 L) and 30% EtOAc/Hexanes (1 L). The filtrate wasconcentrated to give2-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole10 (13.9 g, 100%) as a 20% (wt/wt) solution in ethyl acetate. ¹H NMRshows the presence of about 20% (wt/wt) bis(pinacolato)diboron, and aminor amount (about 10%) of the THP regioisomer,1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole.¹H NMR (400 MHz, CDCl₃) δ 8.37 (s, 1H), 7.62 (dd, J=14 Hz, 2 Hz 1H),7.60 (dd, J=7 Hz, 1 Hz 1H), 7.31 (dd, J=8 Hz, 7 Hz 1H) 5.65 (dd, J=9 Hz,3 Hz 1H) 4.05 (m, 1H) 3.75 (m, 1H) 2.59 (m, 1H) 2.15 (m, 1H) 2.05 (m,1H) 1.75 (m, 3H) 1.34 (s, 12H). LCMS (ESI pos) m/e 245 (M+1).

Example 104-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-2-(2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-4-yl)thieno[3,2-d]pyrimidin-4-yl)morpholine14

To a solution of4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine9 (96.5 g, 223 mmol, 1.0 equiv.) in 1,4-dioxane (1.75 L) was added water(772 ml), sodium carbonate (47.4 g, 447 mmol, 2.0 equiv.) and2-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole10 (73 w/w %, 150.7 g, 325 mmol, 1.5 equiv.). The mixture was degassedfor three times. Bis(triphenylphosphine)palladium (II) chloride (6.28 g,9.94 mmol, 0.04 equiv.) was added and the resulting slurry was degassedfor 4 times. The mixture was heated to 88° C. and stirred for 14 hours.The reaction mixture was cooled to 50° C., concentrated under vacuum tohalf of the total volume and then cooled to 15° C., and acetonitrile(900 ml) was added. After 2 hours of agitation, the resulting slurry wascooled to −5° C., filtered and rinsed with acetonitrile (40 ml), water(90 ml), and acetonitrile (40 ml). The cake was dried in a vacuum ovenat 50° C. for 24 hours to afford a brown-yellow solid (140 g, the Pdcontent: 2000 ppm). The cake was dissolved in methylene chloride (1930ml) and FLORISIL® (60-100 mesh, 193 g, purchased from Aldrich ChemicalCompany, Inc) was then added. The slurry was stirred at the ambienttemperature for a minimum of 5 hours and SILIABOUND®Thiol (28 g) wasadded. The mixture was stirred at ambient temperature for a minimum of12 hours, filtered and rinsed with methylene chloride (2 L), followed bya mixture of methylene chloride (2 L) and ethyl acetate (2 L). All thefiltrate and the rinse were combined and concentrated to give4-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-2-(2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-4-yl)thieno[3,2-d]pyrimidin-4-yl)morpholine14 as an off-white solid (93 g) with Pd content of less than 20 ppm, andcontaining a minor amount of THP regioisomer 14A. ¹H NMR (300 MHz,CDCl₃) δ 1.20-4.30 (br, 8H), 2.61-2.64 (m, 4H), 2.74 (s, 3H), 3.23-3.26(m, 4H), 3.83-3.86 (m, 6H), 3.99-4.02 (m, J=4.15 Hz, 4H), 5.66-5.70 (m,1H), 7.32 (s, 1H), 7.32-7.37 (dd, 8.6 Hz, 7.1 Hz, 1H), 7.77-7.80 (d, 8.6Hz, 1H), 8.22-8.25 (d, 6.99 Hz, 1H), 9.04 (s, 1H). LCMS (ESI pos) m/e598 (M+1).

Example 114-(2-(1H-Indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholineI

4-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-2-(2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-4-yl)thieno[3,2-d]pyrimidin-4-yl)morpholine14 (200 g, 0.3346 mole) was charged to a suitably sized reactor undernitrogen, followed by methanol (3.0 L) and water (0.16 L).Methanesulfonic acid (160.8 g, 1.673 mole, 5.00 equiv.) was slowly addedto the reactor (a mild exotherm is observed). The slurry was stirred atambient temperature for 1 hour, then heated to 65° C. and stirred for 16hours. A sample was taken from the reactor and submitted for HPLCanalysis. HPLC indicated the content of the residual starting materialwas 0.5% (specification<1%). The reaction mixture was cooled to 0-5° C.and stirred for >3 hours, filtered and rinsed with cold methanol (0-5°C., 600 ml). The cake was transferred to a sizable reactor, followed byethyl acetate (1 L) and methyl-tert-butyl ether (2 L). The resultingslurry was stirred at ambient temperature for >4 hours, filtered andrinsed with methyl-tert-butyl ether (200 ml). The cake was dried in avacuum oven at 55° C. for at least 12 hours to afford4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholineI bis mesylate salt as an off-white solid (224 g).

The solid was transferred to a suitably sized reactor followed by theaddition of water (1.34 L). The resulting mixture was heated to 30° C.to obtain a clear solution. The solution was then filtered throughdouble in-line filters (1 micron and 0.45 micron) to remove any foreignmaterial. The filtrate was concentrated at 55° C. under vacuum untilapproximately 80% of the water has been removed. Methanol (3.36 L) wasadded to the reactor through an in-line filter (0.45 micron) and theresulting mixture was cooled to 5° C. and methanesulfonic acid (60.5 g)was slowly added. After stirred at 5° C. for 30 minutes, the mixture washeated to 55° C. and stirred for a minimum of 16 hours. In-process XRPD(X-ray powder diffraction) and DSC (differential scanning calorimetry)confirmed the desired crystalline form and the slurry was cooled to 0°to 5° C. and stirred for a minimum of 3 hours, filtered, and rinsed withcold methanol (0° C. to 5° C., 0.47 L). The cake was transferred to areactor of suitable size, followed by the addition of ethyl acetate (1.0L) and tert-butylmethyl ether (2.0 L). The resulting slurry was stirredat ambient temperature for a minimum of 4 hours, filtered, and rinsedwith tert-butylmethyl ether (1.50 wt). The cake was dried in a vacuumoven at 55° C. for a minimum of 12 hours to afford4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholineI bis mesylate salt as an off-white solid (204 g, 87%). ¹H NMR (300 MHz,DMSO-d₆) δ 2.40 (s, 6H), 3.02 (s, 3H), 3.01 (s, 3H), 3.00-3.87 (br, 8H),3.88-3.89 (m, 4H), 4.10-4.12 (m, 4H), 4.77 (br, 2H), 7.52-7.57 (t, 7.8Hz, 1H), 7.77-7.83 (t, 8.7 Hz, 2H), 8.14-8.16 (d, 7.17 Hz, 1H), 8.76 (s,1H). LCMS (ESI pos) m/e 514 (M+1).

The product I bis mesylate was milled through a jet mill using nitrogenas the process gas. The milling conditions were as follows: Venturipressure: 100 psi; mill pressure: 35 to 50 psi; and feed rate: 3.6 to4.4 kg/hr. The typical recovery is 93% to 97%

Example 12 Thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione 16

A solution of methyl 2-amino-thiophenecarboxylate 15 (95 g) anddichloromethane (2.85 L) was cooled the to −60° C. and chlorosulfonylisocyanate (89.81 g) was added at a rate such that the internaltemperature remains at −60° C. to −55° C. The resulting mixture was thenallowed to warm up to 20° C. and the complete consumption of startingmaterial was confirmed by LCMS. The mixture was then concentrated todryness via rotary evaporator and the solids was stirred in a 5 L flaskwith water (1.9 L) at 75° C. for one hour. The slurry was then cooled to30° C. and a solution of 10 M NaOH (200 mL) was added. The mixture washeated to 85° C. and stirred for 20 min., then cool to room temperature.Conc. HCl was added until pH=1 and the mixture were stirred at ambienttemperature for 18 hrs. The slurry was then filtered and rinsed withcold water (3×300 ml). The cake was dried in vacuum oven at 55° C. for24 hours to afford thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione 16 as anoff-white solid (80.05 g, 79%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.08 (d,J=5.6 Hz, 1H), δ 7.12 (d, J=5.6 Hz, 1H). LCMS (ESI pos) m/e 169 (M+1).LCMS (ESI pos) m/e 169 (M+1).

Example 13 2,4-Dichlorothieno[2,3-d]pyrimidine 17

Phosphorous oxychloride (365 g, 2.38 mol, 5.0 equiv.) was added slowlyto a cold solution of thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione 16 (80 g,0.476 mol, 1.0 equiv.) and N,N-dimethylaniline (42 g, 0.347 mol, 0.75equiv.) in acetonitrile (400 ml) while maintaining the temperature below25° C. The mixture was then heated to 80-85° C. and stirred for 24hours. LCMS indicated that the reaction was complete. The reactionmixture was cooled to 15° C., then poured slowly onto a mixture of iceand cold water (1.0 L). The resulting slurry was filtered, rinsed withcold water (300 ml). The cake was dried in vacuum oven at 40° C. for 24hours to afford dichlorothieno[2,3-d]pyrimidine 17 as an off-white solid(93.4 g, 67% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.62 (d, J=6.4 Hz, 1H),δ 8.16 (d, J=6.4 Hz, 1H). LCMS (ESI pos) m/e 205 (M+1).

Example 14 4-(2-chlorothieno[2,3-d]pyrimidin-4-yl)morpholine 18

Morpholine (85.1, 0.977 mol, 2.2 equiv.) was added to a solution of2,4-dichlorothieno[2,3-d]pyrimidine 17 (91 g, 0.444 mol, 1.0 equiv.).The reaction mixture was stirred at ambient temperature for 1 h and theresulting slurry was filtered, rinsed with water (500 ml). The cake wasdried in vacuum oven at 55° C. for 24 hours to give4-(2-chlorothieno[2,3-d]pyrimidin-4-yl)morpholine 18 as an off-whitesolid (100.3 g, 88% yield).

¹H NMR (400 MHz, DMSO-d₆) δ 3.736 (t, J=4.8 Hz, 4H), δ 3.897 (t, J=5.2Hz, 4H), δ 7.658 (d, J=6.4 Hz, 1H), δ 7.682 (t, J=6.4 Hz, 4H δ). LCMS(ESI pos) m/e 257 (M+1).

Example 15 2-Chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbaldehyde

To a suspension of 4-(2-chlorothieno[2,3-d]pyrimidin-4-yl)morpholine 18(90.2 g, 0.350 mol, 1.0 equiv.) in THF (anhydrous, 1400 ml) at −78° C.was added slowly 2.5 M solution of n-BuLi in hexanes (169 ml, 0.522 mol,1.2 equiv.). The resulting slurry was allowed to warm up to −60° C. anda clear brown solution was observed. The solution was then cooled to−78° C. and DMF (anhydrous, 38.67 g, 0.530 mole, 1.5 equiv.) was addedslowly. The resulting solution was stirred at −78° C. for 0.5 hour, andwarmed up slowly to 0° C. over a period of 1-1.5 hours. The solution wasthen poured slowly to a mixture of 0.25 M aq. hydrochloric acid (3.0 L)and ice water (1.4 L). The resulting slurry was stirred at 0-10° C. for0.5 hour, filtered and rinsed with cold water (0.5 L). The cake wasdried in vacuum oven at 55° C. for 24 hours to afford2-chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbaldehyde 19 as alight yellow solid (90.8 g, 91% yield). ¹H NMR (400 MHz, DMSO-d₆) δ3.778 (t, J=4.8 Hz, 4H), 3.990 (t, J=4.8 Hz, 4H), 8.756 (s, 1H), 10.022(s, 1H). LCMS (ESI pos) m/e 285 (M+1).

Example 164-(2-Chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholine20

To a suspension of2-chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbaldehyde 19 (90.8 g,0.320 mol, 1.0 equiv.), 4-(methylsulfonyl)piperazin-1-ium chloride 8(alternatively named as 1-(methylsulfonyl)piperazine hydrochloride, 92.3g, 0.460 mol, 1.45 equiv.) and sodium acetate (anhydrous powder, 37.7 g,0.460 mol, 1.45 equiv.) in 1,2-dichloroethane (anhydrous, 1.8 L) wasadded trimethyl orthoformate (340 g, 3.20 mol, 10 equiv.). The slurrywas stirred at ambient temperature for at least 6 hours. Sodiumtriacetoxyborohydride (assay>90%, 101.3 g, 0.430 mol, 1.35 equiv.) wasadded and the reaction was stirred for 24 hours. LC/MS indicated thatthe reaction was complete. The reaction was quenched with water (4.4 L)and methylene chloride (4.4 L). The phases were separated and theorganic phase was dried over MgSO₄, filtered and concentrated to give ayellow solid. The crude solid was then stirred in ethyl acetate (2.0 L)at 80° C. for 2 hours. The slurry was cooled to 30-40° C., filtered andrinsed with ethyl acetate (50 ml). The cake was dried in vacuum oven at45° C. to give4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholine20 as an off-white solid (110.2 g, 80% yield). ¹H NMR (400 MHz, DMSO-d₆)δ 2.52-2.55 (m, 4H), 2.89 (s, 3H), 3.12-3.14 (m, 4H), 3.73 (t, J=4.4 Hz,4H), 3.81 (s, 2H), 3.86 (t, J=4.8 Hz, 4H), 7.59 (s, 1H). LCMS (ESI pos)m/e 432 (M+1).

Example 174-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-2-(2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-4-yl)thieno[2,3-d]pyrimidin-4-yl)morpholine21

To a solution of4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholine20 (110 g, 0.250 mol, 1.0 equiv.) in 1,4-dioxane (1.98 L) was addedwater (0.88 L), sodium carbonate (54.1 g, 0.50 mol, 2.0 equiv.) and2-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole10 (50 w/w %, 209.4 g, 0.310 mol, 1.25 equiv.). The mixture was degassedfor three times. Bis(triphenylphosphine)palladium (II) chloride (3.57 g,0.005 mol, 0.02 equiv.) was added and the resulting slurry was degassedfor 4 times. The mixture was heated to 88° C. and stirred for 14 hours.The reaction was complete by LCMS and the reaction mixture was cooled tothe ambient temperature, filtered and rinsed with water (2.0 L).

The crude product was then stirred with FLORISIL® (60-100 mesh, 101 g,purchased from Aldrich Chemical Company, Inc) in methylene chloride (2L) at ambient temperature for at least 5 hours, then filtered, rinsedwith methylene chloride (3 L), followed by a mixture of methylenechloride (2 L) and ethyl acetate (2 L). All the filtrate and the rinsewere combined and concentrated to give a solid (110 g, the Pd content:150 ppm). The solid was dissolved in methylene chloride and SILIABOND®Thiourea (50 g, Silicycle Inc., Crawford Scientific Ltd.) was added. Themixture was stirred for 5 hours, filtered, rinsed with methylenechloride (3 L), followed by a mixture of methylene chloride (2 L) andethyl acetate (2 L). All the filtrate and the rinse were combined andconcentrated to give4-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-2-(2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-4-yl)thieno[2,3-d]pyrimidin-4-yl)morpholine21 as a solid (98 g, Pd content: <10 ppm, 70%) along with a minor amountof THP regioisomer 21A. ¹H NMR (300 MHz, CDCl₃) δ 1.20-2.40 (br, 6H),2.66-2.69 (m, 4H), 2.81 (s, 3H), 3.28-3.31 (m, 4H), 3.78-3.86 (m, 3H),3.91-3.94 (m, J=4.15 Hz, 4H), 3.97-4.00 (m, J=4.21, 4H), 4.15-4.19 (d,1H), 5.74-5.78 (dd, 8.9 Hz, 3.2 Hz, 1H), 7.16 (s, 1H), 7.39-7.44 (dd,8.6 Hz, 7.1 Hz, 1H), 7.84-7.87 (d, 8.7 Hz, 1H), 8.30-8.32 (dd, 7.08 Hz,0.72 Hz, 1H), 9.11 (s, 1H). LCMS (ESI pos) m/e 598 (M+1).

Example 184-(2-(1H-Indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholineII

4-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-2-(2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-4-yl)thieno[2,3-d]pyrimidin-4-yl)morpholine21 (500 g, 0.836 mole) was charged to a sizable reactor, followed bymethanol (9.5 L) and water (250 ml). The resulting slurry was cooled to0° C. and stirred for 0.5 h. A cold solution of concentrated sulfuricacid (H₂SO₄, 5.54 ml, assay 95-98%, 1.004 mole, 1.20 equiv.) and water(250 ml) was slowly added while maintaining the temperature below 10° C.The mixture was allowed to warm up to the ambient temperature andstirred for 20 hours. The slurry was cooled to 5° C., filtered andrinsed with cold methanol (2 L). The cake was charged to a reactor andstirred in a mixture of methanol (9.5 L) and water (25 ml) at 50° C. for3 hours. The slurry was cooled 0-5° C., filtered and rinsed with coldmethanol (2 L). The cake was dried in a vacuum oven at 50° C. for 24hours to afford4-(2-(1H-Indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholineII sulfate salt as a light yellow solid (482 g, 94%). ¹H NMR (300 MHz,DMSO-d₆) δ 3.00-4.00 (br, 8H), 3.01 (s, 3H), 3.85-3.87 (m, 4H),4.00-4.02 (m, 4H), 4.69 (s, 2H), 7.46-7.52 (t, 7.8 Hz, 1H), 7.70-7.73(d, 8.3 Hz, 1H), 7.91 (br, 1H), 8.22-8.25 (d, 7.2 Hz, 1H), 8.82 (s, 1H).LCMS (ESI pos) m/e 514 (M+1).

What is claimed is:
 1. The compound selected from 14 and 14A